Abstract

The ubiquitin proteasome system is responsible for the proteolysis of
important cell cycle and apoptosis-regulatory proteins. In this paper
we report that the dipeptidyl proteasome inhibitor,
phthalimide-(CH2)8CH(cyclopentyl)
CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits
tumor growth of the human lung cancer cell line A-549 in an in
vivo model. In cultured A-549 cells, CEP1612 treatment results in
accumulation of two proteasome natural substrates, the cyclin-dependent
kinase inhibitors p21WAF1 and p27KIP1,
indicating its ability to inhibit proteasome activity in intact cells.
Furthermore, CEP1612 induces apoptosis as evident by caspase-3
activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549
tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days)
resulted in massive induction of apoptosis and significant (68%;
P < 0.05) tumor growth inhibition, as shown by terminal
deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore,
immunostaining of tumor specimens demonstrated in vivo
accumulation of p21WAF1 and p27KIP1 after
CEP1612 treatment. The results suggest that CEP1612 is a promising
candidate for further development as an anticancer drug and demonstrate
the feasibility of using proteasome inhibitors as novel antitumor
agents.

Introduction

The proteasome is a high molecular weight multicatalytic protease
complex. This 20S proteasome (Mr 700,000)
associates with activators to form a 26S proteasome
(Mr 2,000,000), which catalyzes the
ATP-dependent degradation of polyubiquitinated protein substrates
(1,
2,
3)
. The multicatalytic complex has several proteolytic
activities including chymotrypsin-like, trypsin-like,
peptidylglutamylpeptide-hydrolyzing, small neutral amino
acids-preferring, and branched chain amino acid-preferring activities.
Among the proteasome substrates are proteins intimately involved in the
regulation of several important pathways including those of programmed
cell death (apoptosis), cell division cycle, signal transduction, and
antigen presentation (reviewed in
Refs. 4–
8). Aberrant proteasome
activity has been implicated in several pathological conditions such as
cancer, muscular dystrophy, emphysema, leprosy, and Alzheimer’s
disease
(7, 9)
. Of particular interest to us is the
involvement of the proteasome in the degradation of proteins pivotal to
the cell division cycle and apoptosis in human cancer cells. For
example, the degradation by the proteasome of cyclins B and E is
required for exit out of mitosis and entry into the S-phase of the cell
cycle, respectively. The proteasome is also responsible for the
degradation of proteins involved in the regulation of cell
survival/apoptosis (i.e., p53 and nuclear factor-κB) as
well as those involved in the interface between cell cycle and
apoptosis (i.e., the cyclin-dependent kinase inhibitors
p21WAF1 and p27KIP1,
Refs. 4–
8).

The fact that the proteasome has been implicated in many disease states
suggested that proteasome inhibitors have therapeutic potential.
Furthermore, these inhibitors would provide outstanding tools for
determining the role of the proteasome in various physiological
functions. This has prompted many to design, synthesize, and
biologically evaluate proteasome inhibitors.

Several groups have made a variety of structurally unrelated proteasome
inhibitors
(6, 10)
. These include peptide aldehydes such
as PSI
(N-benzyloxycarbonyl-Ile-Glu-(O-t-butyl)-Ala-Leu-H
(11)
or CbzLLnV-H and CbzLLnL-H
(12)
,
which inhibit the chymotrypsin-like activity of the proteasome. Others
have made tripeptide epoxyketones
(13)
, dipeptidyl
boronic acid
(14)
, indanylamide derivatives
(15)
, and vinyl sulfone derivatives
(16)
.

Iqbal et al.(17)
designed a series of
dipeptidyl aldehydes that inhibited potently the chymotrypsin-like
activity of the proteasome. One of these,
CEP1612,
4
was highly selective (>500-fold) for chymotrypsin-like over
trypsin-like activity of the proteasome. CEP1612 was also shown to be
cell permeable and inhibited the proteasome in intact cells with an
IC50 of 1 μm. Initially, CEP1612 was shown to
block MCH-1 antigen processing
(18)
. Subsequently, An
et al.(19)
demonstrated that CEP1612 induces
apoptosis in human prostate, breast, tongue, and brain cancer cell
lines. Furthermore, they demonstrated that CEP1612 induced apoptosis in
SV40-transformed but not the parental normal human fibroblasts WI-38
(19)
. In addition, inhibition of the proteasome activity
is sufficient to overcome Bcl-2- or Bcr-Abl-mediated drug resistance of
tumor cells
(19, 20)
. In this study, human lung
adenocarcinoma A-549 cells were implanted s.c. in nude mice to evaluate
whether CEP1612 can induce apoptosis and inhibit human tumor growth
in vivo.

Materials and Methods

Synthesis of Dipeptidyl Proteasome Inhibitor CEP-1612.

CEP1612 was prepared according to the procedure reported previously
(17)
, except for a key modification in the synthesis of
the NH2-terminal region. Benzyl cyclopentylacetate was
monoalkylated with 1,8-diiodooctane in tetrahydrofuran by sodium
hexamethyldisilazide at −78°C. Substitution with potassium
phthalimide and deprotection of the benzyl ester produced the
NH2-terminal derivative. This was coupled with a
COOH-terminal moiety using an acid chloride activation method. Final
deprotection by 1 n HCl produced the aldehyde, CEP1612.

Cell Culture and Drug Treatment.

Human lung adenocarcinoma A-549 cells were purchased from American Type
Culture Collection (Rockville, MD) and grown in Ham’s F-12K Nutrient
Mix, containing 10% FBS, at 37°C in a humidified incubator
containing 10% CO2. Treatment of cells with the proteasome
inhibitor CEP1612 was performed as described previously
(19)
.

Western Blotting.

Whole-cell extraction and the enhanced chemiluminescence Western blot
assay were performed as we described previously
(21)
.
Briefly, proteins were resolved by 15 or 8% SDS-PAGE gel and
immunoblotted with antibodies against caspase-3 (CPP32),
p21WAF1 (SX118), p27KIP1 (G173-524; PharMingen,
San Diego, CA), and PARP (Boehringer Mannheim). The ECL blotting system
(NEN Life Science Products, Boston, MA) was used for detection of
positive antibody reactions.

Antitumor Activity in the Nude Mouse Tumor Xenograft Model.

Nude mice (Charles River, Wilmington, MA) were maintained in accordance
with the Institutional Animal Care and Use Committee procedures and
guidelines. A-549 cells were harvested, resuspended in PBS, and
injected s.c. into the right and left flank (10 × 106 cells/flank) of 8-week-old female nude mice as reported
previously
(22)
. When tumors reached about 100–150
mm3, animals were dosed i.p. with 0.2 ml once daily.
Control animals received a saline vehicle, whereas treated animals
received injections of CEP1612 (10 mg/kg/day). The tumor volumes were
determined by measuring the length (l) and the width
(w) and calculating the volume (V = lw2/2), as described previously
(22)
.
Statistical significance between control and treated groups was
evaluated by using Student’s t test (P < 0.05).

Effects of CEP1612 on Organ Proteasome Activity.

The ability of CEP1612 to reach mice organs was determined by injecting
nude mice with CEP1612 (10 mg/kg i.p. once) and sacrificing the mice at
times 0, 5, 15, 60, and 120 min after injection. Three mice were
collected per time point. Livers, kidneys, and lungs were then
collected and processed for proteasome activity assay as described
above for tumor proteasome activity measurements.

TUNEL Assay and Immunostaining.

Apoptosis was determined by TUNEL using an in situ cell
death detection kit (Boehringer Mannheim). Frozen sections were
prepared from the treated and untreated tumors. The slides were fixed
in paraformaldehyde (4% in PBS, pH 7.4). After rinsing with PBS and
incubation in permeabilization solution, the tissues were cross reacted
with TUNEL reaction mixture (for 60 min at 37°C in a humidified
chamber), with converter-alkaline phosphatase solution (for 30 min at
37°C in a humidified chamber), and with alkaline phosphatase
substrate solution (Vector Laboratories, Burlington, MA, for 5–10
min). For the immunostaining of p21WAF1 and
p27KIP1, the tumor frozen sections and slides were prepared
as described for TUNEL assay; the anti-p21WAF1 or
anti-p27KIP1 antibody was applied to the slide. The
reactions were analyzed by light microscopy.

Results and Discussion

CEP1612, a cell permeable dipeptidyl aldehyde proteasome
inhibitor, was initially designed and synthesized by Iqbal et
al.(17)
and subsequently shown to block MCH-1
antigen processing
(18)
. More recently, An et
al.(19)
demonstrated that CEP1612 induced apoptosis
in several human cancer cell lines and that normal cells were resistant
to CEP1612-induced apoptosis. In this report, we present evidence for
the ability of CEP1612 to induce the expression of p21WAF1
and p27KIP1 and apoptosis as well as inhibit tumor growth
of the human lung adenocarcinoma A-549 in vivo using the
nude mouse xenograft model.

To determine the in vivo apoptotic and antitumor activity of
CEP1612, we synthesized this molecule as described previously by Iqbal
et al.(17)
, using modifications of the route
as described in “Materials and Methods,” and confirmed its ability
to inhibit proteasome activity (Fig. 1)
⇓
. CEP1612 inhibition of the chymotrypsin-like proteasome activity was
demonstrated using a recombinant 20S proteasome and
Suc-leu-leu-val-Tyr-AMC as substrate as described in “Materials and
Methods.” Fig. 1B⇓
shows that CEP1612 inhibited the
proteasome activity in a concentration-dependent manner with an
IC50 of 60 nm. Our IC50 is higher
than Iqbal et al.(17)
, and this could be
because of the fact that they used human brain 20S proteasomes and
methoxysuccinyl-Glu-Val-Lys-Met-para-nitroanilide as substrate. More
important, however, is the fact that CEP1612 synthesized by us was as
potent as that synthesized by Iqbal et al.(17)
when used in whole cells against the breast carcinoma MCF-7 and
SV40-transformed WI-38 (see below).

A, structure of the dipeptidyl proteasome
inhibitor, CEP1612. B, CEP1612 inhibits the 20S proteasome
activity in vitro. To determine whether the dipeptidyl
proteasome inhibitor CEP1612 blocks proteasome activity in
vitro, a cell-free system was used by incubation of the inhibitor
with a recombinant 20S proteasome from E. coli and
chymotrypsin-like substrate. The proteasome activity was determined as
RFU by measuring with a fluorometer the fluorescence from the AMC
cleaved product as described in “Materials and Methods.”
Data are representative of three independent experiments.
Bars, SD.

We next investigated whether CEP1612 inhibits proteasome activity and
induces apoptosis in cultured A-549 cells. A-549 cells were treated
with CEP1612 (30 μm) for 24 h and lysed, and the
lysates were immunoblotted with antibodies to p21WAF1 and
p27KIP1 as described in “Materials and Methods.”
p21WAF1 and p27KIP1 are two cyclin-dependent
kinase inhibitors that are known natural substrates for the
chymotrypsin-like activity of the proteasome
(23, 24)
.
Fig. 2A⇓
shows that treatment with CEP1612 resulted in the accumulation of both
p21WAF1 and p27KIP1. These data confirmed that
CEP1612 inhibits proteasome activity in A-549 cells. Previously, An
et al.(19)
reported that CEP1612 induces
apoptosis in human breast, prostate, brain, and head and neck cancer
cell lines. To determine whether CEP1612 induces programmed cell death
in A-549 cells, we exposed these cells to CEP1612 and determined its
ability to induce processing and activation of caspase-3 and cleavage
of PARP as described in “Materials and Methods.” Fig. 2B⇓
shows that treatment with CEP1612 resulted in the processing of
caspase-3, as evident from the CEP1612-dependent generation of the p17
form from the CPP32 form. Similarly, CEP1612 treatment also resulted in
the cleavage of PARP to its p85 proteolytic fragment (Fig. 2B)
⇓
. The IC50 of CEP1612 to induce PARP cleavage
in A-549, MCF-7, and SV40-transformed WI-38 was 1 μm and
similar to what An et al.(19)
reported
previously.

The proteasome inhibitor CEP1612 results in accumulation
of cyclin-dependent kinase inhibitors p21WAF1 and
p27KIP1 and induces apoptosis in vitro.A, accumulation of the cyclin-dependent kinase inhibitors
p21WAF1 and p27KIP1 in A-549 cells treated
either with DMSO vehicle or 30 μm CEP1612 for 24 h
is shown. Whole-cell extracts were prepared and analyzed by Western
blot using anti-p21WAF1 and anti-p27KIP1
antibodies. B, activation of caspase-3 and stimulation of
PARP cleavage in A-549 cells treated with 30 μm CEP1612
for 24 h. Immunoblotting was performed using anti-CPP32 and
anti-PARP antibodies. The full-length PARP and
Mr 85,000 fragment (p85/PARP) as well
as pro-CPP32 (CPP32) and activated form
(p17/CPP32) are indicated. Data are representative of two
independent experiments.

The data described above clearly demonstrate that the proteasome
inhibitor CEP1612 induces apoptosis in cultured A-549 cells. To
determine CEP1612 antitumor efficacy and its ability to induce
apoptosis in vivo, we implanted A-549 cells s.c. in nude
mice, and when the tumors were palpable (100–150 mm3), the
mice were either treated with vehicle control or CEP1612 (10 mg/kg/day,
i.p.). After 1 month of daily treatment, the A-549 tumors were removed
and processed for inhibition of proteasome activity and TUNEL as well
as p21WAF1 and p27KIP1 immunostaining as
described in “Materials and Methods.” Fig. 3A⇓
shows that tumors from control animals grew to an average size of
285.3 ± 26.0 mm3. In contrast, tumors from
CEP1612-treated animals grew to an average size of only 176.4 ± 36.6 mm3. Thus, treatment with CEP1612 resulted in
a statistically significant (P < 0.05), 68%
tumor growth inhibition (Fig. 3A)
⇓
. To demonstrate that the
proteasome activity of A-549 tumor cells was inhibited in
vivo, tumor extracts were incubated with purified peptide
substrate for various periods of time, and the chymotrypsin-like
activity of the proteasome was assayed as described in “Materials and
Methods.” Fig. 3B⇓
shows that tumor extracts from control
animals contained proteasome activity that reached 2000 RFUs over 120
min. In contrast, tumor extracts from CEP1612-treated animals contained
only 1050 RFUs, demonstrating that CEP1612 treatment resulted in 43%
inhibition of proteasome activity in vivo (Fig. 3B)
⇓
. To further document that CEP1612 inhibited the
proteasome activity, we next determined by immunostaining whether
treatment of nude mice with this molecule resulted in the accumulation
in the A-549 tumors of two of its natural substrates,
p21WAF1 and p27KIP1. Fig. 4A⇓
shows that tumors from control animals have low levels of
p21WAF1 and p27KIP1. However, tumors from
CEP1612-treated animals contained much higher levels of these
cyclin-dependent kinase inhibitors. Consistent with our in
vitro results of Fig. 2B⇓
, CEP1612 treatment in
vivo was also able to induce accumulation of p21WAF1
and p27KIP1. Thus, CEP1612 injected i.p. was able to reach
the s.c.-implanted A-549 tumors and inhibit the intended target
in vivo. We next determined whether CEP1612 also inhibited
the proteasome activity in mice organs. To this end, we have injected
i.p. 15 nude mice with CEP1612 (10 mg/kg) and sacrificed the mice after
5, 15, 60, and 120 min (3 mice/time point). Livers, lungs, and kidneys
were then processed for proteasome activity assays as described in“
Materials and Methods.” CEP1612 inhibited potently (80%) liver
proteasome activity within 5 min of treatment. In contrast, kidney
proteasome activity was inhibited by only 50% 2 h after CEP1612
injection. Lung proteasome activity was not affected by CEP1612
treatment (Fig. 4C)
⇓
.

A, antitumor efficacy of CEP1612 against human
lung adenocarcinoma A-549 xenografts in nude mouse. A-549 cells were
injected s.c. at day 0 into the right and left flanks (10 × 106 cells/flank) of 8-week-old female nude mice.
When the tumor size reached an average of 125 mm3, animals
(five mice/group) were treated i.p. daily either with vehicle (•) or
CEP1612 (10 mg/kg; ▾) for 31 days, and tumor volumes were determined
as described in “Materials and Methods.” Data are representative of
two independent experiments. Statistical significance between control
and treated groups were evaluated by using Student’s t test
(P < 0.05). Bars, SE.
B, CEP1612 inhibits SE proteasome enzyme activity in
vivo. Nude mice were treated as described in A. At the
end of experiment, tumor specimens were dissected, frozen, and
sectioned. Then tumor extracts from CEP1612 treated (▴), untreated
(▪), or BSA control (•) were incubated with purified
chymotrypsin-like substrates at various times (0–120 min), and the
proteasome activity was determined using a fluorometer as described in“
Materials and Methods.” Data are representative of two independent
experiments; Bars, SD.

CEP1612 accumulates the cyclin-dependent kinase inhibitors
p21WAF1 and p27KIP1 and induces apoptosis
in vivo. A, A-549 xenografts in nude mice were treated and
prepared as described in the Fig. 3
⇓
legend, and immunostaining was
performed by using anti-p21WAF1 and
anti-p27KIP1 antibodies. B, to determine whether
administration of CEP1612 induces apoptosis of A-549 cells in
vivo, nude mice bearing A-549 tumors were treated either with
vehicle (DMSO) or CEP1612 (10 mg/kg/day, i.p.) for 31 days and
sacrificed, and tumor specimens were dissected, frozen, and sectioned;
apoptosis was detected using an in situ TUNEL assay as
described in “Materials and Methods.” Areas of the tumor that are
apoptotic show dark nuclear staining, and p21WAF1 and
p27KIP1 also show intense staining. The fields shown
are × 100. C, to determine whether CEP1612
inhibits the intense proteasome activity in mice organs, we treated the
mice with CEP1612 for various periods of time and collected lungs,
livers, and kidneys and measured their proteasome activity as described
in “Materials and Methods.” Data are representative of two
independent experiments; Bars, SD.

The above results demonstrated that CEP1612 reaches its target and
inhibits human tumor growth in vivo. Whether this inhibition
of tumor growth is attributable to induction of apoptosis was next
determined. Tumor specimens from control and CEP1612-treated animals
were processed for TUNEL assay as described in “Materials and
Methods.” TUNEL analysis demonstrated that A-549 tumors from vehicle
control-treated animals contain no apoptotic cells (Fig. 4B)
⇓
. In contrast, tumor cells from animals treated with
CEP1612 were apoptotic. Thus, CEP1612 treatment resulted in the
induction of massive apoptosis in A-549 tumors in vivo.

Taken together, our data demonstrate that treatment of A-549
tumor-bearing nude mice i.p. with CEP1612 resulted in inhibition of the
proteasome activity in these tumors, accumulation of cyclin-dependent
kinase inhibitors, induction of programmed cell death, and significant
inhibition of tumor growth. However, despite induction of apoptosis, no
tumor regression was observed, suggesting possibly that greater plasma
levels may be required. Alternatively, a subpopulation of the tumor
cells may be resistant to the proteasome inhibitor. Therefore, the
long-term effect on tumors may be tumor growth delay rather than tumor
regression. With regard to side effects, over the 31-day period of
daily i.p. treatment, no overall gross toxicity was observed. Indeed,
animals treated with CEP1612 (10 mg/Kg/day; 31 days) showed no weight
loss, decreased activity, or anorexia. This is consistent with previous
studies from An et al.(19)
that demonstrated
that in cultured cells, human cancer cells were sensitive whereas
normal cells were resistant to CEP1612-induced apoptosis. However, our
pharmacodynamic studies demonstrated that the proteasome inhibitor was
able to reach several organs and inhibit their proteasome activity.
Therefore, more detailed microscopic and macroscopic pathology studies
are required to document the lack of toxicity of this proteasome
inhibitor.

Recently, Orlowski et al.(25)
and Adams
et al.(26)
, using structurally unrelated
molecules, also demonstrated the ability of proteasome inhibitors to
inhibit tumor growth. Orlowski et al.(25)
showed that treatment of Burkitt’s lymphoma-bearing nude mice with
Z-LLF-CHO inhibited tumor growth by 42%. Similarly, Adams et
al.(26)
showed that the dipeptide boronic acid
PS-341 inhibited by 60% the growth in nude mice of the prostate cancer
cell line PC-3. The fact that three structurally unrelated proteasome
inhibitors can inhibit human tumor growth in vivo gives
strong support for proof-of-concept of using proteasome inhibitors as
novel anticancer drugs. The remaining challenge is to design more
potent and selective proteasome inhibitors, with optimal
pharmacokinetic profiles to suppress human tumor growth without
toxicity in clinical settings.

Acknowledgments

We thank the Pathology Core facility at the H. Lee Moffitt
Cancer Center and Research Institute for processing the immunostaining
samples.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 This work was supported in part by research
grants from the National Cancer Insitute (to S. M. S. and A. D. H)
and the National Institute of Aging (to Q. P. D.) H. Lee Moffitt
Cancer Center and Research Institute (to Q. P. D. and S. M. S.).